Chemotherapy using cytotoxic drugs is presently the most commonly used weapon in the treatment of advanced cancer. However, the therapeutic efficacy of cancer chemotherapeutic agents is limited by factors such as tumor heterogeneity, host toxicity, and drug resistance. Recent advances in molecular medicine have provided new opportunities to improve cancer chemotherapy through the development of novel, more selective anti-cancer strategies. The long-range goal of this project is to translate recent advances in our understanding of the role of cytochrome P450 enzymes in cancer pharmacology into improved methods for sensitizing tumor cells to cytochrome P450-activated anti-cancer drugs. This proposal builds on progress made during the last project period in developing a novel, cytochrome P450 prodrug activation-based gene therapy for cancer treatment. The therapeutic potential of this P450 gene therapy derives, in part, from the striking cytotoxic enhancement that is associated with intratumoral, as compared to hepatic P450-catalyzed prodrug activation. This approach is unique among prodrug activation strategies, insofar as it utilizes mammalian genes in combination with proven and tested chemotherapeutic prodrugs currently used in the clinic, rather than novel prodrugs whose therapeutic efficacy and ultimate clinical utility is uncertain. The present application aims to further improve the responsiveness of tumor cells to the widely used, liver P450-activated oxazaphosphorine anti-cancer prodrug cyclophosphamide. Primary emphasis is placed on the elucidation of novel ways to enhance cyclophosphamide therapeutic activity by augmenting bystander cytotoxic responses at the tumor cell level and at the level of tumor-associated endothelial cells. The major aims of this proposal are: 1) to test the hypothesis that tumor cell bystander cytotoxicity, and hence the overall efficacy of P450 gene therapy, can be enhanced by introduction of caspase inhibitors that delay the death of 'P450 factory' tumor cells; and 2) to elucidate the mechanism for the dramatic anti-tumor response that is induced when large, P450-expressing tumors are given cyclophosphamide on a metronomic schedule; these latter studies will investigate the role of a tumor endothelial cell-directed bystander effect in this response, and will determine whether this effect can be enhanced by combination of metronomic cyclophosphamide scheduling with anti-angiogenic therapy. Together, the studies proposed will help elucidate a rational basis for increasing the activity of established anti-cancer prodrugs in a way that moderates toxic host responses and improves therapeutic effects, and will thereby advance the development and implementation of effective prodrug-based therapies for cancer treatment.